In the thymus, central tolerance should eliminate the majority of immature T cells that express autoreactive, pathogenic antigen receptors (??TCRs). However, an unknown number of autoreactive cells escape deletion or commit to immunosuppressive, regulatory linage (Tregs). Tregs control peripheral tolerance and sustain dormancy of potentially autoreactive T cells. However, mice and humans with disabled Tregs rapidly develop multiorgan autoimmunity and die young, manifesting polyclonal activation of almost all CD4+ clones. Thus, we hypothesized that the number of autoreactive clones embedded in the peripheral repertoire can be much higher (i.e. over one-third of all CD4+ cells) as compared to what is currently anticipated. In Specific Aim 1, we will examine how the intrathymic expression of different self-peptides supports or prevents an escape of autoreactive T cells from central tolerance. This approach will document that potentially self-reactive CD4+ clones commonly trespass to lymphoid organs of B6 mice as quiescent cells. Next, we will test these cells ex vivo responses to a known set of self-peptides naturally presented by mouse Ab molecules to determine if these clones are triggered by ubiquitous or specific autoantigens. In Specific Aim 2, we will investigate why mice expressing single autoantigen across the body have main autoimmunity manifestation in specific organs and examine the role of non-classical CD4+ T cells in autoimmunity in this model. In Aim 3 we will use a new single-cell RNA seq system from 10X Genomics, to examine in individual CD4+ cells their transcriptomes and native ??TCRs to identify genes targeted by Tregs in potentially autoreactive cells to keep them dormant and prevent autoimmunity. Overall, this application will revisit the relative importance of various mechanisms of tolerance in the maintenance of homeostasis to self- antigens and can reveal new mechanisms of how Tregs control self-reactivity.